Vascular smooth muscle cells (VSMCs) support nascent blood vessels during early development, but then acquire an advanced differentiated phenotype essential for contraction and blood flow regulation. The major effector of VSMC differentiation is the Serum Response Factor-Myocardin (SRF/MYOCD) transcriptional switch, which binds CArG boxes found in many VSMC-restricted genes. This switch is often compromised in disease states leading to VSMC de-differentiation. While levels of Myocardin are known to be reduced in disease, we know virtually nothing about its regulatory control in vivo. Moreover, the downstream targets of MYOCD (most notably, long noncoding RNAs) are not entirely known. We have generated a number of new mouse models and enabling genomic data that allow us to rapidly define the transcriptional control of Myocd in vivo and elucidate the function of novel SRF-dependent and SRF-independent MYOCD targets. We propose three aims that leverage mouse models with the revolutionary CRISPR-Cas9 genome editing system and state-of-the-art tools in genetics and genomics to test the hypothesis that SRF-dependent and SRF-independent transcription of Myocd and the downstream MYOCARDome function to maintain VSMC homeostasis. Aim 1 will utilize a new, biallelic-tagged Myocd mouse to interrogate candidate enhancers and regulatory elements defined through ChIP-seq, computational prediction, luciferase assay, or circular chromosome conformation capture (4C) assays. Two and three component CRISPR will inform those regulatory regions of critical importance for Myocardin expression. Aim 2 will utilize CRISPR-mediated loss-of-function mice and RNA-seq to begin deciphering the function of two novel genes discovered in screens for MYOCD-inducibility: an SRF-dependent long noncoding RNA gene (Mymsl) and an SRF-independent protein-coding gene (Kank1). Both genes are enriched in VSMC and appear to function in the maintenance of normal VSMC differentiation. ChIP-seq studies will ascertain and validate these MYOCD targets while disclosing the full MYOCARDome in VSMC using the dual epitope- tagged mice of Aim 1. Aim 3 will further characterize the critical regulatory elements (Aim 1) and novel MYOCD target genes (Aim 2) in models of vascular pathobiology (arterial-venous fistula and aortic aneurysm). In addition, we will make use of new loss- and gain-of-function Myocd mice to further advance our understanding of this critical cofactor and its downstream program in relevant models of human disease where the VSMC differentiation program is compromised. Completion of the aims will vertically advance our understanding of the regulatory processes undergirding Myocd expression and the function of novel MYOCD target genes under normal and disease conditions. Such knowledge will inform the next generation of experimental and clinical studies designed to maintain normal levels of Myocardin as a means of thwarting the pervasive de-differentiation of VSMC observed in human disease.